JP2001136993A - Production of ursodeoxycholic ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microbiological production process for ursodeoxycholic ester. SOLUTION: A microorganism capable of producing 7β-hydroxy-steroid dehydrogenase is attached or immobilized onto the surfaces of a hydrophilic immobilized carrier that is charged in an anaerobic interfacial bioreactor and includes the nutrients and water and proliferated to form a layer of the microorganism. The resultant microbial cell layer is brought into contact with the substrate of 7-ketotricholic ester in a hydrophobic organic solvent to effect the biological reduction of 7-ketolithocholic acid thereby producing ursodeoxycholic ester. In this case, a saturation amount of water is added to the hydrophobic organic medium to crystallize out the objective ursodeoxycholic ester on the surface of the microbial layer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to ursodeoxycholic acid, a precursor of ursodeoxycholic acid (hereinafter abbreviated as "UDCA") useful as a solubilizer for cholesterol gallstones and a growth promoter for cultured fish. The present invention relates to a microbial production method of an ester (hereinafter, abbreviated as “UDCA ester”).

[0002]

BACKGROUND OF THE INVENTION UDCA, a secondary bile acid
Is useful as a cholesterol gallstone dissolving agent together with chenodeoxycholic acid (hereinafter abbreviated as "CDCA") [Nakagawa, S., et al. , Lancet, Vol. 2,
367 (1977); Stiehl, A., et al. , Gasteroent
erology, Vol. 57, 1016 (1978)], CDC
A has been pointed out to cause liver damage, and it is considered that UDCA alone is preferable for dissolving gallstones [Fedorowsky, T., et al. , Gastroenterology, Vol.
74, 75 (1978); Macdonald, I .; A., et al. ,
J. Lipid Res., Vol. 22, 458 (1981)].

There is also a report that when UDCA is given to farmed fish, the amylase activity in the digestive tract of the farmed fish increases, thereby improving the digestibility of food and promoting the growth of fish [Mashita Maida et al. , Journal of the Fisheries Society of Japan,
62, 129 (1996)], UDCA is said to be consumed in ton-scale quantities worldwide [Bo
vara, R., et al . Org. Chem., Vol. 58,499
(1993)].

UDCA was previously isolated and purified from the gall bladder of bears [Kurozumi, K., et al. , J. Am. Biochem.,
Vol. 74, 489 (1973)], from the viewpoint of resource depletion due to the capture of bears, it is currently synthesized by an organic synthesis method comprising a multistage reaction. However, the current organic synthesis method has problems such as low yield and the use of metallic sodium or potassium, which has many safety problems [Japanese Patent Publication No. 53-10062;
JP-A-2-13960; JP-B-62-54439; JP-B-63-54720; etc.], and there is a strong demand for the development of a method for producing UDCA which is safer and can be obtained in high yield.

On the other hand, as part of steroid conversion using biocatalysts such as microbial cells and enzymes, cholic acid (hereinafter referred to as "cholic acid") is used.
Oxidation of secondary bile acids such as primary bile acids such as “CA”) or CDCA or lithocholic acid (hereinafter abbreviated as “LCA”) and deoxycholic acid (hereinafter abbreviated as “DCA”); Numerous proposals have been made for methods such as reduction and esterification, and high regioselectivity and stereoselectivity unique to biocatalysis are exhibited. Among these many proposals, the method of synthesizing UDCA includes hydroxylation of methylene group at the 7-position of LCA using a filamentous fungus [Sawada, H., et al.
et al. , Appl. Environ. Microbiol., Vol. 44,12
49 (1082); Kulprecha, S., et al. , Appl. Envi
ron. Microbiol., Vol. 49,338 (1085); JP-B-62-54478; JP-B-62-55840.
Japanese Patent Publication No. 1-37120; Japanese Patent Publication No. 3-29
No. 39], an enzyme-organic synthesis method using regioselective acylation of the 3-hydroxyl group of CA using immobilized lipase as a key step [Sugai, T., et al. , Biosci. Biotech. Biochem.,
Vol. 60, 2059 (1996)], a stereoselective α / β inversion method for the 7-hydroxyl group of CDCA using the intestinal bacterium Clostridium absonanum [Macdonald, I. et al. A. a
nd Roach, P.S. D., Biochem. Biophys. Acta, Vol. 66
5,262 (1981); Macdonald, I .; A., et al. ,
J. Lipid Res., Vol. 22, 458 (1981); Suth
erland, J .; D. and Macdonald. I. A., J. Lipid., Re
s., Vol. 23,726 (1982)], Clostridium absonanum and Bacillus fragilis ( Bacillus f.
ragilis ) and 7α-hydroxysteroid dehydrogenase (hereinafter abbreviated as “7α-HSDH”)-producing bacteria and 7α-hydroxysteroid dehydrogenase such as E.
β-hydroxysteroid dehydrogenase (hereinafter, referred to as
A method of inverting the 7-hydroxyl group of CDCA from the α-configuration to the β-configuration in combination with a producing bacterium [Hirano, H .; and Masuda, N., Lipid Res., Vo
l. 22, 1060 (1981); Macdonald, I .; A., e
t al. , Appl. Environ. Microbiol., Vol. 44,11
87 (1982)].

[0006] All of these known methods have high regioselectivity and stereoselectivity, and extremely high yields have been reported particularly in the oxidation-reduction method using an HSDH-producing strain. (However, the enzyme-organic synthesis method using the lipase is a multi-step reaction, and the yield is not high). However, conversion substrate LCA, CDCA, 7-ketolithocholic acid (hereinafter abbreviated as "7KLCA") or product UDC
A has a strong surface-active effect and destroys the cell membrane of microorganisms even at a very low concentration. Therefore, there is a disadvantage that the addition concentration and the accumulation concentration have to be kept at very low levels.

[0007] Therefore, the present invention can solve all of the problems such as low yield, necessity of multi-step process, danger in production, low substrate concentration and low product accumulation concentration caused by the above-mentioned organic synthesis method. There is a strong need for the development of UDCA synthesis processes.

[0008]

Means for Solving the Problems Accordingly, the present inventor has set forth the following seven points.
The β-hydroxylation of the carbonyl group at the 7-position of the 7KLCA ester using the anaerobic intestinal bacterium Eubacteria aerofaciens having the ability to produce β-HSDH was selected as the target reaction, and this reaction was used to avoid toxicity of hydrophobic poisons. Interfacial bioreactors [Oda, S. et al. and Ohta, H.,
Biosci. Biotech. Biochem., Vol. 56, 1515 (1
992)] to obtain the substrate 7K
It has been found that the LCA ester can be added at a high concentration and that the accumulation of the product UDCA ester can be achieved at a high concentration.

In this conversion system, the bacterium used is an anaerobic bacterium, the solubility of the substrate and the product in organic solvents is low, the expression mechanism of the key enzyme 7β-HSDH is complicated, and the substrate 7KLCA ester is used. And product UDC
There are various problems to be solved, such as the fact that both A esters have extremely strong microbial toxicity, and known interface bioreactors [Oda, S .; and Ohta, H., Biosci. Biot
ech. Biochem., Vol. 56, 2041 (1992); Shinobu Oda and Hiromichi Ota, Journal of the Society of Color Materials, 70, 538 (199)
7); Japanese Patent No. 2542766] cannot be simply applied.

Since the bacterium used in the above reaction is an anaerobic bacterium, it is necessary to completely shut off the inside of the interfacial bioreactor, and for this purpose, the hydrophilic carrier and the substrate-containing hydrophobic It is necessary to make all of the organic solvent layer and the dead space inside the interface bioreactor oxygen-free. Since hydrophobic organic solvents generally have high oxygen solubility, they are particularly effective for microbial oxidation reactions, and the oxidation reactions proceed efficiently even under static conditions [Oda, S., et al . . , J. Ferment. Bioen
g., Vol. 78, 149 (1994); Oda, S., et a .
l . Ferment. Bioeng., Vol. 80, 559 (199
5); Oda, S., et al. , Biosci. Biotech. Biochem.,
Vol. 60, 83 (1996)]. Therefore, when the target reaction of the present invention is applied to an interfacial bioreactor, it is not sufficient to simply degas the gas phase inside the reactor and replace it with gas (for example, nitrogen gas). Also requires degassing-gas replacement.

An example of such an anaerobic interface bioreactor is an anaerobic flat plate interface bioreactor (hereinafter abbreviated as “anaerobic reactor”) as shown in FIG. The anaerobic reactor comprises, for example, a stainless steel reactor base (1) and a transparent vinyl chloride plate (2), both of which are completely adhered by an organic solvent resistant Viton rubber packing (3). In the anaerobic reactor, a hydrophilic carrier (4) represented by a nutrient agar plate is prepared at the bottom of a stainless steel reactor base. The dissolved oxygen in the carrier is degassed and gas-substituted via a gas line (5) before gelation, so that the carrier is made anaerobic, and after gelation, E. coli.
A cell suspension of Aerofacilens is introduced onto the surface of the gelled carrier, and excess water is removed, for example, by blowing nitrogen gas. The transparent vinyl chloride plate (2) is tightly mounted on the reactor base (1) by a clamp (6) via a Viton rubber packing (3), and the gas phase inside the reactor is degassed and replaced with gas. Further, in order to make the anaerobic degree strict and confirm the retention, an oxygen elimination catalyst (7) and an oxygen indicator (8) may be mounted inside the anaerobic reactor in some cases.

The anaerobic reactor having such a configuration is subjected to a preculture for about one day. E. aerofaciens grows in the form of a film, and then the substrate 7
The reduction reaction is started by anaerobically introducing the organic solvent containing the KLCA ester into the reactor. The hydrophobic organic solvent containing the substrate can be easily put into an anaerobic state by, for example, introducing a small number of zeolite and performing degassing and gas replacement.

The 7KLCA ester as a substrate and the UDCA ester as a product in the present invention both have a hydrophobic steroid skeleton and have an α-hydroxyl group at the 3-position, a keto group or β-hydroxyl group at the 7-position and a 24-hydroxyl group. In the present invention, in order to improve the solubility in a hydrophobic organic solvent, 2
The carboxyl group at the 4-position is esterified, specifically methylated, ethylated, propylated or butylated,
Nevertheless, the solubility of the substrate and the product, the UDCA ester, in hydrophobic organic solvents is low, for example, the solubility of 7KLCA methyl ester in dodecane, frequently used in interfacial bioreactors, is only 0.
It is about 2% by weight.

The present inventor has searched for an organic solvent which has a certain degree of polarity and is not so toxic to E. bacteria aerofaciens. As a result, the present inventors have found that medium-chain aliphatic ethers such as dihexyl ether and diisoamyl ether. Can be used as a good reaction solvent. In both cases, the solubility of 7KLCA ethyl as a substrate is about 1% by weight, the solubility of UDCA ester as a product is about 0.7% by weight, and both solvents have low toxicity to E. aerofaciens. In addition, a medium-chain aliphatic ester such as 2-ethylhexyl acetate or dodecyl acetate is used by mixing at an appropriate ratio as a solvent having relatively low toxicity and excellent solubility of the substrate and the product in these solvents. By doing so, it was also confirmed that a higher concentration of 7KLCA ester could be added to accumulate a higher concentration of UDCA ester.

[0015] However, both dihexyl ether and diisoamyl ether are substrates of 7KLCA.
The solubility of the ester is only 1% by weight, and the solubility of the product UDCA ester is as low as only 0.7% by weight. Is virtually impossible. In contrast, acetic acid 2 which is more excellent in the solubility of the substrate and the product
-When a medium chain aliphatic ester such as ethylhexyl or dodecyl acetate is mixed, the solubility of the substrate and the product is improved, but for example, dihexyl ether / acetic acid 2-
2.0% by weight when the mixing ratio of ethylhexyl is 7: 3,
Even at 5: 5, 3.0% by weight of 7KLCA ester can be dissolved. Furthermore, there is a problem that the higher the mixing ratio of the medium-chain aliphatic ester, the greater the toxicity of the medium to microorganisms, resulting in a reduction in reduction rate.

In the present invention, low solubility of the substrate and the product in the reaction solvent is used. That is, by utilizing the relative low solubility of the product in the reaction solvent, the water content in the reaction solvent is increased with the progress of the reduction reaction to further reduce the solubility of the product, and the product is converted into crystals. By excluding from the reaction system, it was found that the UDCA ester as a product can be accumulated at a high concentration, and the present invention has been completed.

Thus, according to the present invention, a microorganism capable of producing 7β-hydroxysteroid dehydrogenase on the surface of a hydrophilic immobilized carrier containing a nutrient source and water, which is filled in an anaerobic interface bioreactor. Is adhered and immobilized, and this is grown to form a microbial membrane. The microbial membrane is brought into contact with a hydrophobic organic solvent in which 7-ketotricholate is dissolved as a substrate to form a microbial membrane.
In producing ursodeoxycholic acid ester by performing a microbiological reduction reaction of ketolithocholic acid ester, by adding a saturated amount of water to the hydrophobic organic solvent, the ursodeoxycholic acid ester is crystallized to form the microorganism. There is provided a method for producing ursodeoxycholic acid ester, wherein the method is deposited on a film surface.

In the present invention, when a 7K LCA ester is reduced to a UDCA ester using an anaerobic interface bioreactor to which E. bacterium aerofaciens is applied, the water content in the reaction solvent is gradually increased as the reduction reaction proceeds. As a result, the maximum solubility of the UDCA ester accumulated in the reaction solvent gradually decreases, and crystallization thereof starts immediately after reaching the saturation concentration. The concentration in the reaction solvent drops sharply due to crystallization of UDCA, but after the water content reaches saturation, the concentration also shows a saturation value, and further, UDCA produced by the progress of the reduction reaction
Crystals corresponding to the amount are deposited on the surface of the carrier, or more precisely, on the surface of the microorganism film grown on the surface of the carrier.

The greatest advantage of the system according to the present invention is that
The problem is that the problem of the solubility of the substrate and the product in the reaction solvent can be solved. Rather, the lower the solubility of the product, the lower the product concentration in the reaction solvent, and as a result, product toxicity to microorganisms can be largely avoided. At this time, in order to overcome the low solubility of the substrate, the substrate may be periodically added by a flow-down method in an amount corresponding to a low concentration.

A mixture with a medium-chain aliphatic ether or a medium-chain aliphatic ester, which is a reaction solvent, has some polar groups (ether group and ester group),
In order to show a water content of 0.1% by weight, trace amounts of water may be added to the reaction solvent until the concentration reaches the limit. At that time, if the amount of water to be added greatly exceeds the limit value, there is a possibility that the microbial membrane formed on the carrier surface may be peeled off due to excessive moisture (water droplets). Therefore, it is often better to embody the effects of the present invention by the water contained in the microbial membrane during the formation of the microbial membrane than by the artificial addition of water, and specifically, to use a normal interface bioreactor. Compared to this, more water, specifically, water that does not cause separation and flow of the microbial membrane can be left in the microbial membrane.

Thus, with the progress of the reduction reaction of 7KLCA ester to UDCA ester, which is carried out by applying E. aerofaciens to an anaerobic interface bioreactor, the UDCA ester generated is successively crystallized as a crystal on the surface of the microorganism membrane. , Resulting in greatly reduced microbial toxicity to microorganisms, resulting in the accumulation of large amounts of UDCA esters.

[0022]

EXAMPLES The present invention will be described more specifically with reference to the following examples. Example ABCM (manufactured by Eiken Chemical Co., Ltd.) adjusted to pH 7.5 was placed on the bottom of a stainless steel reactor (300 × 400 mm) base.
1.0 liter of agar plate (agar content: 2.0% by weight) was placed (plate surface area: 1200 cm ² ). The agar plate contained 0.3% by weight of glucose and 1.0% by weight of mannitol, and was degassed and replaced with nitrogen gas before gelation, and was previously kept in an anaerobic state. After the agar plate has gelled, spread 5 ml of a cell suspension (6 loops / ml) of E. aerofaciens JCM7790 on the plate surface and spray nitrogen gas to prevent the surface gloss derived from moisture from being lost. Excess water was removed. A lid made of a transparent vinyl chloride plate was attached via a Viton rubber packing using a clamp, and the gas phase inside the reactor was degassed and replaced with nitrogen gas. In addition, hydrogen gas was used for one last gas filling. After that, the oxygen removal catalyst (BectonDickin) attached inside the reactor, more precisely inside the lid made of vinyl chloride plate
son Microbiology Systems, Becton Dickinson and Com
pany, USA) was activated and pre-cultured at 37 ° C. for 24 hours to grow and thicken the microbial membrane.

After the pre-culture, 300 ml of a 1% by weight 7KLCA methyl ester dihexyl ether solution was anaerobically injected into the reactor through a butyl rubber plug.
The culture was allowed to stand still at 7 ° C. for 7 days to effect a reduction reaction. The organic solvent layer was sampled daily through a butyl rubber plug using a syringe equipped with a cateran needle, and 7KLCA and UDCA methyl were quantified by gas chromatography.
As a result, the accumulation of UDCA methyl began on day 1, but dropped to 2.5 g / l due to the onset of crystallization on day 4, and remained almost the same thereafter.

After the completion of the reaction, the entire hexyl ether layer was recovered, and then the crystals precipitated on the flat plate surface were recovered as long as they could be picked up. Thereafter, the flat plate surface was repeatedly washed with ethyl acetate to remove the unremoved crystals and the hexyl ether layer. Was recovered. The crystal taken out was dissolved in ethyl acetate, and the bacterial cells attached to the crystal surface were removed by filtration, and then the purity was measured using gas chromatography. as a result,
UDCA-methyl and 7KLCA-methyl in the crystals are about 1
They were mixed at a ratio of 0: 1. Thereafter, the ethyl acetate layer was recrystallized by adding hexane, and the purity was measured by gas chromatography. As a result, the chemical purity of UDCA methyl ester was improved to 99% or more (yield: 48).
%).

On the other hand, the recovered washed ethyl acetate layer was dried over anhydrous sodium sulfate and quantified by gas chromatography. As a result, UDCA methyl and 7KLCA methyl were mixed at a ratio of about 10: 2. Ethyl acetate
Recrystallization of hexane increased the chemical purity of UDCA methyl to 98% (11% yield). Further, the recovered hexyl ether layer was dried with anhydrous sodium sulfate,
After removing hexyl ether by silica gel column chromatography, acetone-hexane was used to remove hexyl ether.
KLCA methyl and UDCA methyl were fractionated, and the resulting U
The DCA fraction was recrystallized using ethyl acetate-hexane,
Purified to a chemical purity of 98% (16% yield). Thereafter, the collected crystals of all UDCA methyl were hydrolyzed with alkali (at 90 ° C. for 3 hours), and the crystals recovered by acid precipitation were recrystallized from methanol-water and ethyl acetate-hexane. Purity was measured by high performance liquid chromatography. As a result, the overall yield was 77% (provided that the conversion yield was 94% when corrected for the residual amount of the substrate 7KLCA methyl), and the chemical purity was 99% or more.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view of an example of an anaerobic plate-type interfacial bioreactor.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Reactor base made of stainless steel 2 Transparent vinyl chloride plate 3 Packing made of viton rubber 4 Hydrophilic immobilization carrier 5 Gas line 6 Clamp 7 Oxygen elimination catalyst 8 Oxygen indicator

Claims (3)

[Claims] 1. A microorganism capable of producing 7β-hydroxysteroid dehydrogenase is attached and immobilized on the surface of a hydrophilic immobilization carrier containing a nutrient source and water filled in an anaerobic interface bioreactor, This is grown to form a microbial membrane, and the microbial membrane is contacted with a hydrophobic organic solvent in which 7-ketritocholate is dissolved as a substrate to carry out a microbial reduction reaction of 7-ketotricholate. In producing ursodeoxycholate, a saturated amount of water is added to the hydrophobic organic solvent to precipitate ursodeoxycholate as crystals on the surface of the microorganism membrane. A method for producing an acid ester. 2. The microorganism according to claim 1, wherein the microorganism is Eubacterium aerofaciens.
The manufacturing method as described. 3. A medium chain aliphatic ether such as dihexyl ether or diamyl ether, wherein the hydrophobic organic solvent is
3. The method according to claim 1, wherein the medium-chain aliphatic ether is mixed with a medium-chain aliphatic ester such as 2-ethylhexyl acetate or dodecyl acetate.